Neutrophils, key effector cells in microbial killing and host defense, contain in their azurophil granules preformed polypeptides with potent antimicrobial activity in vitro. The long term goal of the research described in this proposal is to determine the molecular basis for the function of two of these antimicrobial proteins: azurocidin and p29b. Results from our group, as well as others, indicate that the protein we identified on the basis of its antimicrobial activity, p29b is most likely identical to a novel elastase-like protease, proteinase 3, and to the Wegener's granulomatosis autoantigen, hence will be referred to as proteinase 3 (PR3). The proteolytic function of PR3 will be examined in addition to its antimicrobial activity. Binding of azurocidin and PR3 to whole bacteria and cell envelope components will be measured. The effects of both antimicrobial proteins on the physiological functions of E. coli will be determined at the level of outer membrane, cytoplasmic membrane and macromolecular synthesis. A full-length cDNA for azurocidin will be isolated and the complete nucleotide sequence will be determined. To facilitate the studies on the mode of action of these proteins, recombinant forms of azurocidin and PR3 will be produced, using bacterial, insect cell/baculovirus or mammalian expression systems. Native and recombinant antimicrobial proteins will be used for the analysis of functional sites of azurocidin and PR3. Chemical modification and mutational analysis will be employed. The altered proteins will be characterized with respect to protease activity, binding to bacteria and antimicrobial activity.